Project Summary/Abstract Listeria monocytogenes is facultative intracellular food-borne pathogen that provides an extremely amenable model for basic studies on host-pathogen interactions. This proposal is based on the results of a genetic screen that revealed a critical role of bacterial redox sensing for the upregulation of virulence gene expression in vivo. We found that L. monocytogenes glutathione synthase was upregulated during infection and determined that glutathione is the allosteric activator of the major virulence transcription factor PrfA. Based on these findings, and after decades of investigation by multiple groups, we successfully recapitulated intracellular virulence gene expression by simply adding reducing agents to bacteria grown in a chemically defined synthetic media. This finding led to another genetic screen to identify bacterial mutants that were either more sensitive or resistant to the growth inhibitory property of high concentrations of reducing agents. Analysis of the resistant mutants that arose from this screen led to the discovery that L. monocytogenes possess a Flavin-based Extracellular Electron Transport chain (FLEET) that can transport over 100,000 electrons/bacterium/second to flavin moieties present on two extracellular lipoproteins, PplA and FrdA, which can transfer electrons to ferric iron and fumarate via activation of FrdA, which encodes a fumarate reductase. We show that FLEET can mediated anaerobic growth using either ferric iron or fumarate as electron acceptors. Mutants that were more sensitive to reducing agents clustered in perR, a redox-sensing transcription factor that controls the transcriptional response to oxidative stress. We hypothesize that the oxidative stress is being generated by FLEET that we show is producing superoxide in the presence of oxygen. FLEET orthologues were found in 100s of Firmicute species including pathogens and members of the microbiota and we propose that it represents a versatile electron transfer hub present in diverse Gram-positive bacteria, that provides a selective advantage to bacteria growing anaerobically in the intestine. We also propose that FLEET activity has other consequences aerobically that may have profound effects on the cell biology of infection, virulence, and immunity.